Calcium Binding by Synaptotagmin’s C2A Domain is an Essential Element of the Electrostatic Switch that Triggers Synchronous Synaptic Transmission
Published Date:Jan 25 2012
Source:J Neurosci. 32(4):1253-1260.
Keywords:Amino Acid Sequence
Animals, Genetically Modified
Molecular Sequence Data
Protein Structure, Tertiary
Pubmed Central ID:PMC3567453
Funding:MN-61876/MN/OMHHE CDC HHS/United States
NS-045865/NS/NINDS NIH HHS/United States
R01 NS045865-05/NS/NINDS NIH HHS/United States
Howard Hughes Medical Institute/United States
Description:Synaptotagmin is the major calcium sensor for fast synaptic transmission that requires the synchronous fusion of synaptic vesicles. Synaptotagmin contains two calcium-binding domains: C2A and C2B. Mutation of a positively charged residue (R233Q in rat) showed that Ca2+-dependent interactions between the C2A domain and membranes play a role in the electrostatic switch that initiates fusion. Surprisingly, aspartate-to-asparagine mutations in C2A that inhibit Ca2+ binding support efficient synaptic transmission, suggesting that Ca2+ binding by C2A is not required for triggering synchronous fusion. Based on a structural analysis, we generated a novel mutation of a single Ca2+-binding residue in C2A (D229E in Drosophila) that inhibited Ca2+ binding but maintained the negative charge of the pocket. This C2A aspartate-to-glutamate mutation resulted in ∼80% decrease in synchronous transmitter release and a decrease in the apparent Ca2+ affinity of release. Previous aspartate-to-asparagine mutations in C2A partially mimicked Ca2+ binding by decreasing the negative charge of the pocket. We now show that the major function of Ca2+ binding to C2A is to neutralize the negative charge of the pocket, thereby unleashing the fusion-stimulating activity of synaptotagmin. Our results demonstrate that Ca2+ binding by C2A is a critical component of the electrostatic switch that triggers synchronous fusion. Thus, Ca2+ binding by C2B is necessary and sufficient to regulate the precise timing required for coupling vesicle fusion to Ca2+ influx, but Ca2+ binding by both C2 domains is required to flip the electrostatic switch that triggers efficient synchronous synaptic transmission.
text/plain image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg
You May Also Like: